A. Field of the Invention
The present invention relates to insect adipokinetic hormone signal sequences useful for promoting the processing and secretion of foreign proteins in insect systems.
B. Description of the Related Art
Baculovirus expression vectors (BEVs) have become extremely important tools for the expression of foreign genes, both for basic research and for the production of proteins with direct clinical applications in human and veterinary medicine (W. Doerfler, Curr. Top. Microbiol. Immunol., 131:51-68 (1968); V. A. Luckow and M. D. Summers, Bio/Technology, 6:47-55 (1988a); L. K. Miller, Annual Review of Microbiol., 42:177-199 (1988); M. D. Summers, Curr. Communications in Molecular Biology, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1988)). BEVs are recombinant insect viruses in which the coding sequence for a chosen foreign gene has been inserted behind a baculovirus promoter in place of the viral gene, e.g., polyhedrin (G. E. Smith and M. D. Summers, U.S. Pat. No., 4,745,051, which is incorporated herein by reference).
Thus, baculoviruses have gained popularity as expression vectors because of the advantages presented above. The BEV system is currently being employed in over 700 laboratories for the overexpression and production of many different gene products. To date, more than 50 different genes have now been expressed by employing this system (V. A. Luckow and M. D. Summers, Bio/Technology, 6:47-55 (1988)).
The use of baculovirus vectors relies upon the host cells being derived from insects. The proteins expressed by the BEVs are, therefore, synthesized, modified and transported in host cells derived from insects. Most of the genes that have been inserted and produced in the baculovirus expression vector system have been derived from mammalian species.
At present, the only mode of achieving secretion of a foreign gene product in insect cells is by way of the foreign gene's native signal peptide. Because the foreign genes are usually from non-insect organisms, their signal sequences may be poorly recognized by insect cells, and hence, levels of expression may be suboptimal.
Heretofore, the efficiency of expression of foreign gene products seems to depend primarily on the characteristics of the foreign protein. On average, nuclear localized or non-structural proteins are most highly expressed, secreted proteins are intermediate, and integral membrane proteins are the least expressed. One factor generally affecting the efficiency of the production of foreign gene products in a heterologous host system is the presence of native signal sequences (also termed presequences, targeting signals, or leader sequences) associated with the foreign gene. The signal sequence is generally coded by a DNA sequence immediately following (5' to 3') the translation start site of the desired foreign gene.
The expression dependence on the type of signal sequence associated with a gene product can be represented by the following example: If a foreign gene is inserted at a site downstream from the translational start site of the baculovirus polyhedrin gene so as to produce a fusion protein (containing the N-terminus of the polyhedrin structural gene), the fused gene is highly expressed. But less expression is achieved when a foreign gene is inserted in a baculovirus expression vector immediately following the transcriptional start site and totally replacing the polyhedrin structural gene. Insertions into the region -50 to -1 significantly alter (reduce) steady state transcription which, in turn, reduces translation of the foreign gene product. Use of the pVL941 vector optimizes transcription of foreign genes to the level of the polyhedrin gene transcription. Even though the transcription of a foreign gene may be optional, optimal translation may vary because of several factors involving processing: signal peptide recognition, mRNA and ribosome binding, glycosylation, disulfide bond formation, sugar processing, oligomerization, for example.
The properties of the insect signal peptide are expected to be more optimal for the efficiency of the translation process in insect cells than those from vertebrate proteins. This phenomenon can generally be explained by the fact that proteins secreted from cells are synthesized as precursor molecules containing hydrophobic N-terminal signal peptides. The signal peptides direct transport of the select protein to its target membrane and are then cleaved by a peptidase on the membrane, such as the endoplasmic reticulum, when the protein passes through it.
While it has been recognized that the signal sequence associated with a foreign inserted mammalian gene is recognized by the insect cell system and cleaved at the correct sites, the present invention is premised on the use of an insect signal sequence instead of or together with the mammalian signal sequence so as to further enhance the expression of the foreign gene in the insect cell system.